Rheumatoid anemia is typically multifactorial and related to the chronic inflammatory nature of the disease, and includes iron deficiency, mainly due to therapy-induced gastrointestinal blood loss. The detection of iron deficiency in a population with anemia of chronic disease (ACD) is of clinical relevance, since 1) iron deficiency anemia (IDA) is treatable, 2) the diagnosis may prelude the need for further investigations into the cause of the anemia, and 3) it may prevent unnecessary prescription of iron supplementation.
Unfortunately, diagnosis of iron deficiency with currently available laboratory parameters is hampered by the lack of a gold standard and is even more complex and nonspecific when concomitant inflammatory conditions are present (1, 2). Proinflammatory stimuli contribute to ACD directly by inhibition of erythropoiesis and indirectly by decreasing the iron available for heme synthesis (3). The latter may be attributed to inflammation-induced increased levels of the iron regulatory peptide hepcidin. Elevated hepcidin levels reduce intestinal iron absorption as well as iron release from macrophages through interaction, internalization, and degradation of the cellular iron exporter ferroportin, resulting in iron sequestration in the reticuloendothelial system (4, 5). Consequently, the total body iron content is normal, but less iron is available for erythropoiesis. In contrast, in IDA, in which there is an absolute iron deficiency, hepcidin is suppressed, which leads to induction of iron absorption from the gut. Since hepcidin has been shown to be differently affected by inflammation and iron deficiency, it has been advocated as a potential biomarker to assess iron deficiency in patients with inflammatory conditions (6–9).
The hemoglobin (Hgb) contents of erythrocytes (red blood cells [RBCs]) and of reticulocytes (RBC-Hgb and Ret-Hgb) have also been explored as biomarkers for diagnosing iron deficiency (10–13). Ret-Hgb and RBC-Hgb reflect the hemoglobinization of the earliest-released reticulocytes, which circulate for only 1–2 days, and that of mature erythrocytes, respectively (14). Reduced Ret-Hgb and RBC-Hgb values indicate that the iron supply for the bone marrow is too low to allow normal hemoglobinization (12). Since these parameters are not directly affected by inflammation but are functional measures of iron availability, they might be of relevance to detect iron deficiency in conditions of chronic inflammation, such as rheumatoid arthritis (RA) (11). To assess the added diagnostic value of measuring serum and urine hepcidin, Ret-Hgb, and RBC-Hgb (measured as reticulocyte Hgb equivalents [Ret-He] and RBC Hgb equivalents [RBC-He], respectively), for the detection of iron deficiency in patients with anemia and inflammation, we explored their diagnostic characteristics and compared them with conventional indices of iron status in a cross-sectional study of 106 outpatients with RA.
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In this cross-sectional exploratory study of RA patients attending an outpatient clinic, serum and urine hepcidin levels and hemoglobin content parameters were assessed in an effort to identify markers of iron deficiency anemia in patients with inflammatory disease. The detection of absolute iron deficiency in RA patients with anemia is a clinical challenge, since conventional iron status screening parameters, e.g., transferrin, iron, and ferritin, are influenced by acute-phase responses (23). TfR reflects iron stores linearly to the degree of iron demand of erythroblasts, but inflammatory stimuli may suppress its concentrations by inhibition of erythropoietic activity (2, 24). The sTfR index was developed to overcome this effect (15) and has gained merit in diagnostic algorithms together with Ret-Hgb (1, 8, 12); however, each of these markers has its own disadvantages in predicting iron deficiency (2, 23–25). Figure 3 provides an overview of serum iron parameters and hemoglobin content parameters in patients with RA and anemia.
Figure 3. Overview of indicators of serum iron status in rheumatoid arthritis patients with ACD and in those with IDA. A, ACD. In states of inflammation, increased hepcidin quantities are released into the circulation by hepatocytes (36, 37). Hepcidin binds, internalizes, and degrades the iron exporter ferroportin (FPN), which results in a decrease in iron export from enterocytes and macrophages into the bloodstream (38). As a consequence, serum iron and transferrin saturation (TSAT) decrease while iron stored within macrophages increases, leading to elevated serum ferritin levels. The inflammation-induced suppression of erythropoiesis counteracts the decreased availability of iron (3, 24).Therefore, the serum (or soluble) transferrin receptor (sTfR) concentration and the hemoglobin content of reticulocytes and erythrocytes may remain relatively normal (3, 24, 33, 39) (see also Figure 1). B, IDA. Iron deficiency reduces hepcidin (even in the presence of inflammation ), which results in maximal release of iron from enterocytes and macrophages into the circulation. Nevertheless, ferritin and iron levels and TSAT decrease. Consequently, the limited iron supply to the bone marrow will increase the sTfR concentration to allow maximal iron uptake by erythroblasts. Ret-Hgb and RBC-Hgb levels are reduced, indicating insufficient iron availability for erythropoiesis (10, 31). RES = reticuloendothelial system (see Figure 1 for other definitions).
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In our RA patients, serum hepcidin levels correlated highly with urine hepcidin and ferritin levels and the sTfR index, but not with hemoglobin content parameters. Moreover, in the comparison of categories of anemia as defined by conventional parameters, serum hepcidin performed well as a diagnostic test of iron deficiency, including when inflammation was present, and discriminated both the IDA group and the combined IDA/ACD group from patients with ACD. Urine hepcidin, RBC-Hgb, and Ret-Hgb also enabled differentiation between iron deficiency and ACD, albeit with lower statistical significance compared to serum hepcidin.
Hepcidin levels below 2.4 nmoles/liter discriminated IDA and IDA/ACD patients from ACD patients with RA. Apparently, the induction of hepcidin by inflammation is blunted by low iron stores. This suppression of hepcidin in anemic patients with iron deficiency and with inflammation is corroborated by findings of animal studies (26) and in various other patient groups, i.e., in a mixed population of patients with chronic infection, autoimmune disease, or malignancy (20, 27), in cancer patients with inflammation (6), and in patients with acute inflammation in an intensive care unit (28). In contrast, in another heterogeneous population of patients with chronic inflammation and anemia, hepcidin levels did not differentiate patients with IDA/ACD from those with ACD (8); this may be attributed to the use of a different stratification scheme than was used in the present study.
Since hepcidin concentrations at the lowest level of detection (<0.5 nmoles/liter) were indicative of IDA, we explored the added value of urine hepcidin as a more sensitive marker in the lower concentration range. Although measuring urine hepcidin instead of serum hepcidin did not add to the level of diagnostic accuracy in the current and previous studies (18), it might be an attractive alternative in the developing world, as a noninvasive tool for assessing iron deficiency as a safety measure before starting oral iron administration programs (29).
Our findings enable us to propose a novel model for selecting RA patients who would likely benefit from iron supplementation (Figure 4). Since rheumatoid anemia is heterogeneous, more than one single parameter might be necessary to allow diagnosis of iron deficiency. We reasoned that patients with inflammation (test 1) and a hepcidin level of <2.4 nmoles/liter (test 2) will have iron deficiency, while hepcidin levels ≥7.6 nmoles/liter are diagnostic for ACD. Patients whose hepcidin levels fell between 2.4 and 7.6 nmoles/liter were further characterized using Ret-Hgb (test 3), with levels of <1.9 fmoles being indicative of concomitant iron deficiency. For IDA, a hepcidin level of <0.5 nmoles/liter was highly specific. Using these definitions to assess the need for iron supplementation, all patients from the combined IDA/ACD group would benefit from such supplements, with no ACD patients receiving this treatment unnecessarily. This model needs to be confirmed in other populations and may benefit from further international standardization of hepcidin measurements (30).
Figure 4. Proposed algorithm for assessing which rheumatoid arthritis patients with anemia may benefit from iron treatment, based on test characteristics described in the text. CRP = C-reactive protein; ESR = erythrocyte sedimentation rate (see Figure 1 for other definitions).
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The study also provides insights regarding hepcidin and hemoglobin content levels in IDA and ACD patients in comparison to control RA patients without anemia. Both Ret-Hgb and RBC-Hgb discriminated IDA patients from non-anemic RA controls. These observations are consistent with previous reports of convincing ROC characteristics demonstrating the ability of these indices to detect iron deficiency in otherwise healthy subjects and patients undergoing dialysis (10, 14, 31, 32). Interestingly, in RA patients with ACD, we found that levels of hepcidin and the hemoglobin content parameters did not differ significantly from those obtained in non-anemic patients. It appears that in RA patients with chronic disease activity, hepcidin concentrations do not increase to levels seen in acute inflammation. In accordance with the current concept of a causal role of hepcidin in iron-restricted erythropoiesis, the absence of significantly increased hepcidin levels in the ACD group likely explains the similarities in hemoglobin content parameters between the ACD and the non-anemic control RA patients. Moreover, the RA control group differs from a healthy population in terms of, e.g., the presence of inflammatory markers and/or other indices of disease activity.
There are very few reported studies comparing hemoglobin content parameters in healthy controls with those in patients with ACD. In a heterogeneous cohort of ACD patients, the mean cell hemoglobin level was similar to that in age-matched controls, despite elevated hepcidin levels (27). In another mixed group of patients with ACD, levels of hemoglobin content parameters were normal as compared to reference values (8). A similar study showed a nonsignificant trend toward reduced Ret-Hgb concentrations in patients with inflammation; no hepcidin data were available (33). Interestingly, increased hepcidin levels do not necessarily lead to reduced hemoglobin content, despite the induction of iron withholding by hepcidin. Another mechanism related to ferroportin expression on reticulocytes is probably involved (34, 35). Clearly, more and larger studies are needed to further assess levels of hepcidin in patients with chronic inflammation and its association with parameters of hemoglobin content.
To our knowledge, this is the first study of both hepcidin and hemoglobin content parameters in an RA cohort. In the absence of a gold standard, we were dependent on conventional iron parameters to classify the anemia groups IDA, IDA/ACD, and ACD. Notably, this classification scheme precludes direct comparison of hepcidin or hemoglobin content parameters with any of the conventional parameters used for detection of true iron deficiency in RA patients. We believe the strength of this study lies in its unbiased cross-sectional inclusion of RA patients, and its limitation lies in the small numbers of patients in the various anemia categories.
What lessons can be learned from this study? Is it time for the rheumatologist to add hepcidin and hemoglobin content parameters to the clinical measurements that are performed? The present results clearly demonstrate the potential utility of serum hepcidin, and to lesser extent Ret-Hgb and RBC-Hgb, in the detection of iron deficiency in RA patients with inflammation and anemia. Our findings indicate that these novel parameters can provide added value in diagnosing iron deficiency in patients with rheumatoid anemia; larger studies are needed to confirm this.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Swinkels had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Van Santen, van Dongen-Lases, de Vegt, van Riel, van Ede, Swinkels.
Acquisition of data. Van Dongen-Lases, Laarakkers, van Riel, van Ede, Swinkels.
Analysis and interpretation of data. Van Santen, van Dongen-Lases, de Vegt, Laarakkers, van Ede, Swinkels.